Phosphatidylserine externalization in caveolae inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in ECV304.
It has been evidenced that plasma membrane Ca(2+)-ATPase (PMCA) is localized at caveolae. However, the caveolar function of PMCA in living cells has never been demonstrated. In the present study, PMCA is exclusively localized at caveolae from ECV 304 cells demonstrated by sucrose gradient fractionation and the co-localization of PMCA with caveolin-1 was visualized by confocal microscopy. We found that PMCA is the main mechanism involved in Ca(2+) efflux in ECV 304 cells. Treatment of cells with MbetaCD to disrupt caveolae significantly reduced the Ca(2+) efflux, and the rate of decay is 4.45+/-0.14 min(-1) in the absence of MbetaCD and 1.99+/-0.038 min(-1) in the presence of MbetaCD. Moreover, the replenishment of cholesterol restored the reduction of the PMCA-mediated Ca(2+) efflux in the presence of MbetaCD. Consistent with Ca(2+) efflux in living cells, the activity of the reconstituted PMCA in membranes extracted from cells in vitro was decreased in the presence of MbetaCD. It was found that phosphatidylserine, which is normally in the inner leaflet of plasma membranes and is able to stimulate PMCA was relatively enriched in caveolae. Importantly, the treatment of cells with MbetaCD concomitantly increased the phosphatidylserine externalization. Taken together, our results suggest that activation of PMCA in caveolae is modulated by phosphatidylserine, and phosphatidylserine externalization induced by MbetaCD reduced the interaction of phosphatidylserine with PMCA, subsequently PMCA-mediated Ca(2+) efflux in ECV 304 cells.
Zhang
Jie
J
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
Xiao
Ping
P
Zhang
Xujia
X
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D021941:Caveolae / Q000187:drug effects / Q000201:enzymology*; D051242:Caveolin 1 / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000172:deficiency; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D005089:Exocytosis / Q000187:drug effects; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D013109:Sphingomyelins / Q000378:metabolism; D034421:Sus scrofa; D047392:beta-Cyclodextrins / Q000494:pharmacology
D051242:Caveolin 1; D008081:Liposomes; D010718:Phosphatidylserines; D013109:Sphingomyelins; D047392:beta-Cyclodextrins; D002784:Cholesterol; D053499:Plasma Membrane Calcium-Transporting ATPases; C031215:betadex